Phosphatidylinositol (PI)-3 kinases are a family of enzymes implicated in the control of diverse cellular processes, including membrane protein trafficking, cell migration and apoptosis. The goal of this proposal is to examine the mechanism(s) of action of PI-3 kinases on membrane protein trafficking. A protein has been identified that binds tightly to PI(3)P, but not to other phosphorylated inositides such as PI(4)P, PI(4,5)P2, nor to other acidic phospholipids. This protein is termed EEA1, and contains motifs similar to those found in yeast proteins implicated in trafficking to the vacuole. Preliminary results suggest that EEA1 may be a necessary element in insulin-regulated GLUT4 trafficking in the endosomal system. We propose four specific aims: 1) To test the hypothesis that the RING finger defines the kinetics, specificity and structural basis for the binding of EEA1 to 3' phosphoinositides. 2) To test the hypothesis that EEA1 function is a necessary element for insulin action on GLUT trafficking. 3) To characterize the structure and general function of accessory peptides that have been found to interact with EEA1. 4) To determine which of the known PI-3 kinases present in mammalian cells is relevant for EEA1 function.